Role of Individual eNOS Phosphorylation Sites in Regulation of eNOS Activity in Endothelial Cells

Abstract

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO). Protein phosphorylation is one of the important mechanisms for regulation of eNOS at the posttranslational level. Effects of Ser/Thr phosphorylation on eNOS conformation and activity are due to introduction of a negative charge at a specific site. Our lab’s previous work utilized Ser or Thr to Asp mutation to mimick eNOS phosphorylation, and wild-type and mutant forms of eNOS proteins were expressed and purified from a baculovirus/Sf9 cell system. To further determine the role of individual eNOS phosphorylation sites in regulation of eNOS activity in endothelial cells, in this study, we mutated all of five sites of eNOS phosphorylation to Asp or Ala. After infection of bovine aortic endothelial cells with recombinant eNOS adenoviruses, we measured eNOS activity and NO release using a cGMP reporter cell assay. We show that mimicking phosphorylation at Ser116 decreases eNOS activity compared with wild-type eNOS. Mimicking phosphorylation at Ser635 and Ser1179 at the same time does not increase eNOS activity to a greater extent than mimicking phosphorylation of either alone. In addition, removal of any of the five Ser/Thr phosphorylation sites does not affect thapsigargin- or vascular endothelial growth factor-stimulated NO release, demonstrating that maximal agonist-stimulated NO release does not require phosphorylation of any single eNOS phosphorylation site. (Supported by NIH and AHA).